Dmem f12 medium

The human choriocarcinoma‐derived cell line BeWo was obtained from ATCC (Burlington, ON, Canada) and cultured as described previously.20, 21 Briefly, cells were cultured in DMEM/F12 medium supplemented with 10% charcoal‐stripped foetal bovine serum (WISENT Inc. Quebec, Ca), 100 IU/mL of penicillin and 100 μg/mL of streptomycin at 8% O₂ (5% CO₂, 37^οC) (Invitrogen Canada Inc., Burlington, ON, Canada). Cells were seeded (4 × 10⁴ per well, respectively) in 6‐well plates and cultured for 24 hour at 8% O₂ (5% CO₂, 37°C). Syncytialization of BeWo cells was induced by treatment with forskolin (25 μmol/L; Sigma‐Aldrich) for 72 hour and subsequently these cells were treated with DEX (10⁻⁶ mol/L) or vehicle for a further 72 hour at 2% O₂ (5% CO₂, 37°C). Non‐syncytialized BeWo cells (control) were treated with DEX (10⁻⁶ mol/L) or vehicle for 72 hour at 2% O₂. After treatments, cells were then collected and stored at −80°C for total RNA and protein extraction.

We used the CRISPR/Cas9 system to establish a stable BRCA1-KO in human fibroblasts as previously described [14 (link)]. Cells were maintained as per supplier’s recommendations. When cells reached 30% confluence, they were treated with DMEM-F12 medium (Wisent, Saint-Bruno, Canada) supplemented with antibiotics and 10% cancer patient sera or control sera, which had been filtered through 0.2 μm filters. Otherwise, cells were exposed to an exosome load of 25–40 μg/ml (which corresponded to 2.6–4.1e + 07 particles/ml of culture medium. Cells were maintained in these conditions at 37 °C in humidified atmosphere containing 95% air and 5% CO₂ with medium change every second day for 3 weeks. When cells reached 80–90% confluence, they were passaged 1 in 6 using 0.05% Trypsin-EDTA (Wisent, Saint-Bruno, Canada). To confirm that there was no contamination or carry-over of cells from human serum, aliquots of the culture medium were placed in a culture plate and incubated at 37 °C, 5% CO₂ for 4 weeks.

All MCF10A-derived cell lines were maintained in standard MCF10A growth medium without antibiotics (DMEM/F12 medium (Wisent Bioproducts, 319-075-CL), 5% horse serum (Gemini Bio, 100508), 10 µg ml⁻¹ insulin (Thermo Fisher Scientific/Gibco, A11382II), 0.5 mg ml⁻¹ hydrocortisone (Sigma-Aldrich, H4001), 20 ng ml⁻¹ EGF (R&D Systems, 236-EG-01M) and 100 ng ml⁻¹ cholera toxin (List Biological Laboratories, 100B)). MCF10A stably expressing doxycycline-inducible HA-AKT2-WT and HA-AKT2-E17K (pTRIPZ constructs) were maintained in 0.5 μg ml⁻¹ puromycin. NIH-3T3 mouse fibroblasts were grown in DMEM (Wisent Bioproducts, 319-005-CL) with 10% heat-inactivated FBS (Thermo Fisher Scientific, 10438026). SUM159, MDA-MB-468 and T47D cell lines were maintained in RPMI 1640 (Wisent Bioproducts, 350-000-CL) with 10% heat-inactivated FBS. Cell lines stably expressing PANK2 or PANK4 variant constructs in pLenti-Ubc-IRES-Neo (pLUIN) were maintained in 250 µg ml⁻¹ G418 (Geneticin; neomycin analogue) (Invivogen, ant-gn-1). All of the cell lines were grown at 37 °C under 5% CO₂ and high humidity, and were typically trypsinized and split every 2 days, keeping cell densities subconfluent. Cells were confirmed to be negative for mycoplasma contamination using the MycoAlert Detection Kit (Lonza, LT07-218).

Kidneys from P5 Vangl2^Δ/CD and Cre+;Vangl2^+/+ mice were dissected out, cut in half and pelvic/medullary zones were separated from cortices. The separated tissues were enzymatically dispersed for 2 hours at 37°C in DMEM/F12 medium (Wisent, Saint-Bruno, QC, Canada), supplemented with 0.22% Collagenase (C0130, Sigma-Aldrich, Oakville, ON, Canada), 30 μg/ml DNase (D4263, Sigma-Aldrich, Oakville, ON, Canada), 1% Penicillin-Streptomycin (P/S, Thermofisher Scientific, Waltham, MA, USA), 2.5 mg/ml Dispase II (D4693, Sigma-Aldrich, Oakville, ON, Canada) and 10% Fetal Bovine Serum, FBS (Wisent Inc., Saint-Bruno, QC, Canada), washed in Phosphate Buffered Saline, PBS, pH7.4, and plated on the collagen-coated plates in collecting duct growth medium made up of DMEM adjusted to an osmolarity of 600 mOSM, glucose, NaCl and urea, supplemented with non-essential amino-acids (NEAA, 1%), ITS (1%), mouse Epidermal Growth Factor, EGF (10ng/ml, TonboBiosciences, San Diego, CA), hydrocortisone (50picoM/mL, Sigma-Aldrich), HEPES (1mM), P/S (1%) and 2% FBS. After 5 days in culture, cells were harvested and cell pellets were stored at -80°C until use.

HK2 and mPTCs were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States), and cultured in Dulbecco’s modified Eagle medium (DMEM) and DMEM/F-12 medium (319-075-CL, Wisent, Canada), respectively. The medium was supplemented with 10% (v/v) fetal bovine serum (FBS) (FBSSA500-S, AusGeneX, Molendinar, Australia). All the cells were maintained in a humidified atmosphere of 5% CO₂ at 37^°C.

Human FLS were derived from synovial tissue collected during open surgery of patients diagnosed with RA or osteoarthritis (OA), or from healthy controls (H). Patients were recruited in collaboration with Dr. Frédéric Balg. The RA patients fulfilled the American College for Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria for RA classification [1 (link)]. FLS were isolated according to standard procedures [2 (link)] and were cultured in DMEM-F12 medium (Wisent, StBruno, Qc, Canada) supplemented with 10% FBS (Gibco BRL, Burlington, ON, Canada) and 40 mg/mL gentamycin (Wisent, St-Bruno, QC, Canada). Cells were used between passages 3 and 8 [3 (link)]. The study was approved by the Centre Hospitalier Universitaire de Sherbrooke Ethics committee, and written consent was obtained from all participants, protocol number 07-113.

DMEM-F12 medium and newborn bovine serum were purchased from Wisent Corporation (Wisent, Canada). Immortalized mouse proximal tubular cells (mPTCs) from Sciencell Research Laboratory (Cat #: M4100) were maintained at 37°C with 5% CO₂. The cells were subcultured using 0.25% trypsin-0.02% EDTA at 70–80% confluence (Invitrogen, USA). Cells were treated with TGF-β1 at a concentration of 10 ng/mL to induce phenotypic transition with or without a pretreatment of MnTBAP (1.14 μM).